William C. Faquin, MD, PhD Director, Head & Neck Pathology Professor of Pathology Massachusetts Eye & Ear Massachusetts General Hospital Harvard Medical School
THE MILAN SYSTEM AND MOLECULAR ADVANCES IN THE CYTOLOGIC DIAGNOSIS OF SALIVARY GLAND TUMORS Salivary Gland FNA
Even if you do not adopt the Milan System in your practice, reviewing the structure of a reporting system will provide insight to salivary gland FNA! Salivary gland tumors are one of the most heterogeneous groups of neoplasms – So what role is there for FNA? Salivary Gland FNA- Pleomorphic Adenoma
4 Salivary Gland FNA – Pleomorphic Adenoma
5 Salivary Gland FNA- Adenoid Cystic Carcinoma
6 Salivary Gland FNA- Solid Adenoid Cystic Carcinoma
7 SALIVARY GLAND FNA: How Far Can We Go?
Usually Specific Diagnosis Sometimes Specific Diagnosis Usually Descriptive Diagnosis
Pleomorphic adenoma Adenoid cystic carcinoma Basal cell adenoma, tubulotrabecular and solid types Warthin tumor LG mucoepidermoid carcinoma HG mucoepidermoid carcinoma Acute and chronic sialadenitis Carcinoma ex PA Salivary duct carcinoma Basal cell adenoma, Metastasis Polymorphous low-grade membranous type adenocarcinoma Reactive lymph node Small cell carcinoma Basal cell adenocarcinoma Lymphoma Mucocele Epithelial-myoepithelial carcinoma Lymphoepithelial cyst Oncocytoma MASC LESA Acinic cell carcinoma SOME SALIVARY GLAND FACTS
• Tumors: • 0.4-13.5 per 100,000 people (uncommon) • Older adults, females, parotid gland • Approx. 75% are benign • Risk of malignancy is inversely proportional to the size of the gland (20% in parotid; 80-89% in oral cavity)
SALIVARY GLAND FNA
Part of the reason for the high accuracy: A majority of SG neoplasms are pleomorphic adenomas, Warthin tumor, or metastatic cancer
Effectiveness of Cytomorphology alone: » Sensitivity: 86-100% » Accuracy: » Benign/low grade vs HG malignant: 81-98% » Specific lesion: 48-94%
SALIVARY GLAND FNA
Rationale for FNA: –Guide the clinical management/pre-op strategy: » Non-neoplastic Clinical follow-up » Benign tumor/low-grade carcinoma Limited resection » Metastatic disease to parotid LNs LN resection » Lymphoma Heme-Onc referral » High-grade primary carcinoma Radical resection Reporting System for Salivary Gland FNA
Why do we need a new reporting system for salivary gland cytology? Salivary Gland FNA
Diagnostic Terminology • Current reporting confusion: – Diversity of diagnostic categories, vs. – Descriptive reports (no categories), vs. – Surgical pathology terminology • General agreement on the need for a defined set of diagnostic categories for salivary gland FNA – Clarity of communication (implicit cancer risk) – Exchange of data across institutions • The Milan System for Reporting Salivary Gland Cytopathology The Milan System for Reporting Salivary Gland Cytopathology
• Sponsored by the ASC and the IAC • Over 40 participants from 14 countries • Goal is to produce a practical classification system that is user-friendly and internationally accepted. • The system will be evidence-based. • Print Atlas available in 2017 • Web-Based Atlas will also be available through the ASC Participants: 40+ Members from 14 Countries Cytopathologists, Surgical Pathologists, Molecular Pathologists, ENT Surgeons
• 1. Overview of Diagnostic Terminology and Reporting: – Core group, Bruce Wenig, Raja Seethala, Andrew Field, Nora Katabi • • 2.Non-diagnostic: – Mariapia Foschini (lead), Laszlo Vass, Esther Diana Rossi, Jhala Nirag, Philippe Vielh, Kayoko Higuchi, , Ivana Kholova, Makato Urano • • 3. Non-neoplastic: – Bill Faquin (lead), Massimo Bongiovanni, Sule Canberk, Marc Pusztaszeri, Tarik Elsheik, Dan Kurtycz, Fabiano Callegari, Oscar Lin • • 4. AUS: – Marc Pusztaszeri (lead), Zubair Baloch, Bill Faquin, Diana Rossi • • 5. Neoplastic (benign & SUMP): – Zubair Baloch (lead), Jeff Krane, Lester Layfield, Marc Pusztaszeri, Jerzey Klijanienko, Ritu Nayar, Celeste Powers, Pinar Firat, Guido Fadda • • 6.Suspicious for malignancy: – Diana Rossi (lead), Syed Ali, Ashish Chandra, Zarha Maleki, Bo Ping, He Wang, Andrew Field, Yun Gong, • • 7.Malignant: – Güliz Barkan (lead), He Wang, Philippe Vielh, Stefan E. Pambuccian, Swati Mehrotra, Mousa Al-Abbadi, Eva Wojcik • • 8. Ancillary Studies: – Mark Pusztaszeri (lead), Jorge Reis-Filho, Fernando Schmitt, Raja Seethala • • 9. Clinical Management: .. – Mark Varvares (lead –MGH), Piero Nicolai (Italy), Mandeep Bajwa The Milan System for Reporting Salivary Gland Cytopathology Diagnostic Categories
• 1) Non-Diagnostic • 2) Non-Neoplastic • 3) Atypia of undetermined significance • 4) Neoplastic: – a) Benign – b) Uncertain malignant potential • 5) Suspicious for Malignancy • 6) Malignant
The Milan System for Reporting Salivary Gland Cytopathology
The ROM will depend upon the nature of the Specimen and the salivary gland site.
18 Non-Diagnostic
• Insufficient quantitative and/or qualitative cellular material to make a cytologic diagnosis. • 10% would be a target maximum rate • Includes aspirates with benign elements only • Includes non-mucinous cyst contents Non-Diagnostic: Benign salivary gland elements only NORMAL SALIVARY GLAND FNA
For aspirates containing only normal salivary gland elements, a cautionary note is recommended. Note: Clinical and radiologic correlations are recommended to ensure that the aspirate is representative of the lesion; the findings in this aspirate do not explain the presence of a salivary gland mass. Non-Diagnostic: Non-mucinous cyst contents DDX: Ductal cyst, pseudocyst, cystic neoplasm
Absence of an epithelial component Non-Neoplastic
• Specimens lacking evidence of a neoplastic process: • Inflammatory, metaplastic, and reactive (includes acute, chronic, and granulomatous sialadenitis, sialadenosis, etc…) • Reactive lymph nodes (flow cytometry is needed) •Clinico-radiological correlation is essential to ensure that the specimen is representative of the lesion. Non-Neoplastic: Reactive Lymph Node Mixed population of lymphs, Tingible body macrophages, germ center frags REACTIVE PROCESS VS LYMPHOMA
• IMMUNOPHENOTYPING combined with cytomorphology is the key to diagnosing and subtyping reactive conditions vs lymphoma. For negative lymph nodes, caution is warranted: A note suggesting repeat FNA or tissue biopsy if lymphadenopathy persists. Non-Neoplastic: Chronic Sialadenitis
Hypocellular, cohesive basaloid groups, inflammation Non-Neoplastic: Granulomatous Sialadenitis
DDX includes infection, sarcoidosis, and neoplasm Non-Neoplastic: Sialolithiasis Atypia of Undetermined Significance (AUS)
• Cannot entirely exclude a neoplasm. • Heterogeneous category • A majority will be reactive atypia or poorly sampled neoplasms. • Specimens are often compromised (eg, air-drying, blood clot). • Should be used rarely (<10 % of all salivary gland FNAs).
Atypia of Undetermined Significance (AUS): Mucinous Cyst Contents Only- Cannot exclude MEC Atypia of Undetermined Significance (AUS): Oncocytic metaplasia vs Neoplasm Atypia of Undetermined Significance (AUS): Reactive vs basaloid neoplasm
Sclerosing polycystic adenosis Neoplasm
i) Benign Neoplasm: Reserved for clear-cut benign neoplasms This category will include classic cases of PA, WT, lipoma, etc… ii) Salivary Gland Neoplasm of Uncertain Malignant Potential: Diagnostic of a neoplasm; however, a diagnosis of a specific entity cannot be made. A malignant neoplasm cannot be excluded. Neoplastic: Benign Warthin Tumor
Oncocytes, chronic inflammation, and cystic debris Neoplastic: Benign Pleomorphic Adenoma
Matrix-rich types of PA are the easiest. Neoplastic: SUMP Pleomorphic Adenoma With Atypia or Metaplasia
Squamous metaplasia Focal atypia Neoplastic: SUMP Myoepithelioma Neoplastic: SUMP Basal Cell Neoplasm- DDX basal cell adenoma, cellular PA, AdCC Suspicious for Malignancy
• Aspirates which are highly suggestive of malignancy but not definitive. • Often high grade carcinomas with limited sampling or other limitation
Suspicious for Malignancy AdCC with Artifact Malignant
• Aspirates which are diagnostic of malignancy. • Sub-classify into specific types and grades of carcinoma: e.g. low grade vs high grade. • "Other" malignancies such as lymphomas, sarcomas and metastases are also included in this category and should be specifically designated. Malignant
Salivary Duct Carcinoma Undifferentiated Carcinoma Malignant: Adenoid Cystic Carcinoma Malignant: Mucoepidermoid Carcinoma Malignant: HG B-Cell Lymphoma Salivary Gland FNA
• Improvements in IHC and molecular testing will make the Milan System and salivary gland FNA in general more effective. • It is critical that the FNA specimen include adequate material for ancillary studies in difficult cases. Ancillary Studies to Improve the FNA Diagnosis of Head and Neck Tumors
Immunocytochemistry LBP Smears Cell block FISH RT-PCR Next Generation Sequencing
NGS-SNaPshot Panel at MGH
• Anchored Multiplex PCR (AMP)
• ~190 target amplicons across 39 genes and 50+ rearrangements
• High-quality sequence: - Staggered start sites - >100X target coverage - Molecular indexing - Bi-template coverage - ~2% analytical sensitivity
• Fast turn-around (~2 weeks)
• Cost-effective (<$500)
• Small tissue amounts (5-10 ng)
NORMAL SALIVARY GLAND Immunohistochemical Markers
SOX10
Ker 7, 19, CAM 5.2, EMA Ker 7, CAM 5.2, DOG1
Ker 5/6, S-100, p63, calponin, SM actin Ker 5/6, p63 Increasing Availability of Molecular Markers For Salivary Gland Tumors
Mammary analogue secretory carcinoma: ETV6-NTRK3; t(12:15) Pleomorphic adenoma & Ca ex PA: PLAG1; t(3;8) HMGA2 rearrangement Clear cell carcinoma: EWSR1-ATF1; t(12:22) Mucoepidermoid carcinoma: MECT1/MAML2; t(11:19) Cribriform Adenocarcinoma: PRKD rearrangement Adenoid cystic carcinoma: MYB-NFIB; t(6:9) Basal cell adenoma: CTNNB1 mutations
IHC and Molecular Analysis Applied to Selected Salivary Gland Tumors Pleomorphic Adenoma Diagnostic Problems Arise from Variants of PA
• Cellular PA with sparse matrix
• Focal adenoid cystic–like areas
• Cytologic atypia
• Metaplasia – Squamous
– Mucinous
– Sebaceous
– Oncocytic Pleomorphic Adenoma with Mucinous Metaplasia: Can be difficult to distinguish from MEC Pleomorphic Adenoma Immunohistochemistry:
• Markers of both ductal and myoepithelial cells: • Keratin 7, CEA, EMA, SOX10
– Myoepithelial markers:
• Smooth muscle actin
• Calponin
• S-100
• Keratin 5/6
• P63
• GFAP Pleomorphic Adenoma
Cytogenetics: • PLAG1 rearrangements (50-60%) – Present in PAs in different tissues – Nuclear localization – Functions to activate transcription (Zn finger) • HMGA2 rearrangements (10%)
http://www.uniprot.org/uniprot/Q6DJT9 PLAG-1 Immunoreactivity: Overexpressed in 94% of PA Does not distinguish benign from malignant
Contributed by Dr. J. Krane, BWH Carcinoma Ex Pleomorphic Adenoma: Most cases have PLAG1 or HMGA2 Rearrangements
Salivary duct carcinoma ex PA Adenoid Cystic Carcinoma Adenoid Cystic Carcinoma: Classic Cribriform Pattern Adenoid Cystic Carcinoma Immunohistochemistry:
• Positive for keratin 7, CEA, EMA
• Positive for myoepithelial markers: – Smooth muscle actin
– Calponin
– S-100
– Keratin 5/6
– P63
• SOX10+
• CD117 (KIT) +
• MYB + Adenoid Cystic Carcinoma
CD117 Immunohistochemistry:
• Over 90% are strongly positive for CD117 (KIT)
• Protein overexpression but no mutation identified
• Useful for all variants including solid forms
• Unusual IHC pattern Immunoreactivity for CD117 (KIT) in AdCC:
Tubular Type Solid Type
Luminal Cells + MYB immunostaining is a useful ancillary test for distinguishing adenoid cystic carcinoma from pleomorphic adenoma in FNA specimens
FNA BIOPSY Adenoid Cystic Carcinoma: Recent Advance – MYB Translocation Cytogenetics: • t(6:9) MYB oncogene-NFIB transcription factor • In salivary gland, this finding by FISH is specific for AdCC
FISH contributed by Dr. Joaquin Garcia, Mayo Clinic Basal Cell Adenoma/Adenocarcinoma Basal Cell Adenocarcinoma – Cytologically indistinguishable from adenoma Basal Cell Adenoma & Adenocarcinoma
Immunohistochemistry:
• Positive for keratin 7, CEA, EMA
• Positive for myoepithelial markers
• Nuclear beta-catenin + (esp. common in adenoma) Basal Cell Adenoma & Adenocarcinoma • CTNNB1 mutation – 3p21 • Beta-Catenin overexpression – Present at cell junctions – Part of WNT signaling pathway Nuclear Beta-Catenin in Basal Cell Adenoma Jo et al. AJSP 2016;40:1143-1150. Mammary Analogue Secretory Carcinoma FNA of Mammary Analogue Secretory Carcinoma MASC Immunohistochemistry:
• Positive for: – S-100
– Mammaglobin
– Keratin 7, 8, 19
– EMA
– GCDFP-15
– MUC1, MUC4
• Negative for: – Myoepithelial markers (p63 etc)
– Androgen receptor
– DOG1 +/- MASC: Immunohistochemical Studies
GCDFP-15+ S-100+
Mammoglobin+ Mammary Analogue Secretory Carcinoma: Cytogenetics: For difficult cases, test for rearrangement
ETV6-NTRK3 rearrangement:
T(12:15)(p13;q25)
MASC is only known salivary gland primary
Detected on histology or cytology using:
FISH
RT-PCR
Next-Gen Sequencing
FISH Contributed by Dr. Joaquin Garcia, Mayo Clinic Acinic Cell Carcinoma Acinic Cell Carcinoma DOG1 and Acinic Cell Carcinoma
Discovered on GIST
Calcium-activated chloride channel protein
Strong diffuse staining seen in most acinic cell carcinomas SOX-10 and Acinic Cell Carcinoma SRY-related HMG-box 10 (SOX10) protein
Transcription factor: neural crest formation, and maintenance of Schwann cells and melanocytes.
Acinar cells and intercalated ducts
Diffuse strong nuclear staining seen in most acinic cell carcinomas SOX10 and Salivary Gland Tumors POSITIVE
Acinic cell carcinoma
Adenoid cystic carcinoma
Epithelial-myoepithelial carcinoma
Myoepithelial carcinoma
Pleomorphic adenoma
NEGATIVE
Salivary duct carcinoma
Mucoepidermoid carcinoma
Oncocytoma
Warthin tumor MUCOEPIDERMOID CARCINOMA Low Grade Mucoepidermoid Carcinoma: Hypocellular with few groups of bland epidermoid cells; Common cause of FN FNA Mucoepidermoid Carcinoma: IHC is non-specific Immunohistochemistry:
• Positive for: – Keratin 5,6,7,8,19
– EMA
– CEA
– p63
• Negative for: – SM actin
– Calponin
– S-100
– SOX10 Mucoepidermoid Carcinoma: Can be useful for difficult cases
Cytogenetics: • t(11:19) translocation • MECT1/MAML2 • FISH or NGS • More common in low grade • Seethala et al Am J Surg Pathol (2010) – 60% overall – LG 74%, IG 63%, HG 32% – Useful in small biopsies
FISH contributed by Dr. Joaquin Garcia, Mayo Clinic SUMMARY
• Salivary gland cytology presents many diagnostic challenges • The Milan System for Reporting Salivary Gland Cytoplathology will help to produce a more uniform diagnostic structure – Improved communication between treating clinician and pathologist – Improved patient care • Availability of IHC and molecular markers can greatly improve the accuracy of salivary gland FNA! The Milan System for Reporting Salivary Gland Cytopathology
We look forward to implementing the Milan system – Thank You!